Glutatione S-transferase Research Articles

Abstract 885: GSTM1 CNV and promoter SNPs are associated with REC/SPT and survival after Retinoid Acid treatment for head and neck cancer

Abstract Purpose: This study sought to determine if copy number variant (CNV) combined with other genetic variants in the glutatione S-transferase Mu class1 (GSTM1) promoter have an important impact on recurrence and overall survival in head and neck cancer patients after retinoid acid treatment. Experimental Method: This study included 441 head and neck patients treated with retinoid acid. Among these cases, 133 experienced recurrence of disease and/or a second primary tumor, while 308 have single primary disease. TaqMan real-time PCR and direct sequencing were used to determine genetic variants in GSTM1. Recurrence and secondary primary tumor (REC/SPT) were evaluated by logistic regression modeling and overall survival was estimated by Kaplan-Meier analysis using STATA. Results: Regression analysis revealed significantly decreased REC/SPT and increased overall survival in subjects with at least 2 copies of GSTM1 (OR=0.57; 95%CI=0.35-0.95) but not in those with 1 copy of the gene. Kaplan-Meier survival analysis also revealed longer overall survival in patients with at least 2 copies of GSTM1. Allele frequency analysis showed −498 C>G, −426 A>G, −339 C>T polymorphisms were significantly associated with REC/SPT in head and neck cancer patients a(OR 0.12 (0.02-0.87), 0.30 (0.11-0.80) and 1.87 (1.00-3.51), respectively. Further survival analysis demonstrated that the −498G, −426G and −339C alleles significantly increased the overall survival in head and neck cancer subjects. Conclusion: These results suggest the GSTM1 CNV combined with single nucleotide polymorphisms is a better predictor of REC/SPT and overall survival of head and neck cancer after retinoic acid treatment than just measuring the presence/absence of GSTM1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 885.

Homocysteine Thiolactone Induces Cardiac Dysfunction: Role of Oxidative Stress

This study investigates the cardiac functioning in male Wistar rats after treatments with methionine and homocysteine thiolactone (HcyT). The rats were distributed into 3 groups and treated for 8 weeks. Group I was the control (CO) group, given water, group II was treated with methionine, and group III with HcyT (100 mg/kg). Morphometric and functional cardiac parameters were evaluated by echocardiography. Superoxide dismutase (SOD), catalase, and glutathione S-transferase activities, chemiluminescence, thiobarbituric acid reactive substances, and immunocontent were measured in the myocardium. Hyperhomocysteinemiawas observed in rats submitted to the both treatments. The results showed diastolic function was compromised in HcyT group, seen by the increase of E/A (peak velocity of early (E) and late (A) diastolic filling) ratio, decrease in deceleration time of E wave and left ventricular isovolumic relaxation time. Myocardial performance index was increased in HcyT group and was found associated with increased SOD immunocontent. HcyT group demonstrated an increase in SOD, catalase, and glutatione S-transferase activity, and chemiluminescence and thiobarbituric acid reactive substances. Overall, these results indicated that HcyT induces a cardiac dysfunction and could be associated with oxidative stress increase in the myocardium.

Anticancer activity of fungal taxol derived from Botryodiplodia theobromae Pat., an endophytic fungus, against 7, 12 dimethyl benz(a)anthracene (DMBA)-induced mammary gland carcinogenesis in Sprague dawley rats

Breast cancer is the second most prevalent cancer worldwide and their incidence increases gradually. Taxol (paclitaxel), a potent anticancer drug, is naturally isolated from the bark of the Pacific yew. Taxol is widely used in the treatment of ovarian, lung and breast cancer. The increased demand for taxol, coupled with its limited availability from the protected Pacific yew, has had researchers scrambling for alternate sources. The purpose of the present study is to investigate chemopreventive effect of fungal taxol derived from a novel endophytic fungus Botryodiplodia theobromae Pat., isolated from a medicinal plant Morinda citrifolia Linn. The fungal taxol is found to be active against the 7, 12 dimethyl benz(a)anthracene (DMBA)-induced mammary gland carcinogenesis in Sprague dawley rats. The enzymic and non-enzymic antioxidants i.e. superoxide dismutase (SOD), catalase (CAT), glutatione peroxidase (GPx), glutatione-S-transferase (GST), reduced glutathione (GSH), vitamin C and vitamin E were evaluated in control and experimental groups. Lipid peroxides levels (LPO) were also tested. Histological analysis of breast tissue was analyzed by haematoxylin and eosin staining to assess the cytoprotective role of fungal taxol active against breast cancer. Immunohistochemical analyses were also performed to evaluate the effect of fungal taxol on the inflammatory marker such as Cyclooxygenase-2 (COX-2) in control and experimental groups. The results showed that the fungal taxol significantly suppresses the DMBA-induced breast cancer in Sprague dawley rats.

The use of biochemical responses to assess ecotoxicological effects of Pharmaceutical and Personal Care Products (PPCPs) after injection in the mussel Elliptio complanata

A biomarker approach was undertaken using the mussel Elliptio complanata to assess the ecotoxicological effects after injection of a range concentration (0–10 mM) of three different PPCPs: carbamazepine, caffeine, methotrexate; and an effluent extract (C8) from St. Lawrence wastewaters treatment plant (Montreal, Canada). A battery of biomarkers, involving oxidative stress and genotoxicity responses: glutation-S-transferase (GST), ethoxyresorufin O-deethylase (EROD), dibenzylflourescein dealkylase (DBF), xanthine oxidoreductase (XOR) activities, lipid peroxidation (LPO) and DNA damage were determined in gonad and digestive gland tissues after 48 h of injection. Results showed an induction of the oxidative metabolism with increasing pharmaceutical concentration in those mussels injected with the PPCPs and the effluent extract. Phase I detoxification enzymes were significantly induced ( p < 0.05), concretely DBF activity was significantly induced after caffeine, carbamazipine and C8 injection; and EROD activity after C8 and methotrexate injection. Oxidative stress induction only lead to lipid peroxidation ( p < 0.05) in organisms injected with carbamazepine and caffeine and DNA damage in organisms injected with methotrexate ( p < 0.05). EROD and DBF enzymatic activities have been found to be suitable biomarkers to determine bioavailability of pharmaceuticals. LPO and DNA damage to determine possible associated adverse effects. Nevertheless, their validation in realistic exposure scenarios and under exposure conditions should be performed in future research.

Association of Glutatione S-Transferase (GSTM1 and GSTT1) Gene Deletions in Korean Patients with Alcoholism

Alcoholism is caused by a complex interaction between genetic and environmental factors. Findings obtained from several studies indicate that some tissue damage occurring in alcohol abusers is due to the generation of reactive oxygen species during the ethanol metabolism The objective of this study was to examine the associations between the polymorphisms of glutathione S-transferase (GST) M1 and T1 genes and Korean male patients with alcoholism. We investigated the distribution of deletion of GSTM1 and GSTT1 in Korean male patients diagnosed with alcoholism (n=133) and Korean male control subject without alcoholism (n=91) with polymerase chain reaction (PCR) method. GSTM1 showed significant associations with alcoholism susceptibility (p=0.0002). But GSTT1 showed no significant associations (p=0.0948). In combined analysis, both gene deletion and GSTM1 deletion were associated with alcoholism (p ＜0.0001 and p＜0.0150). These results suggest that GSTM1 gene deletion might play an important role in risk for alcoholism.

Expression profiling of senescent-associated genes in human dermis from young and old donors. Proof-of-concept study

It is often described that it is difficult to really discriminate the cause of intrinsic skin aging. The aim of this study was to compare the profiles of expression of senescence-associated genes in biopsies of dermis from young and old human donors. TGF-beta1 was up-regulated in the dermis of old donors as well as the TGF-beta1-regulated genes. The anti-oxidant enzymes Selenium-dependent Glutathione peroxidase and Glutatione S-Transferase Theta 1 were also up-regulated in old dermis as well as Tumor Necrosis Factor Receptor Superfamily 1A. None of these genes had altered expression level in skin fibroblasts embedded in a collagen matrix and exposed to sublethal doses of UVB, suggesting their involvement in intrinsic aging. This study represents a proof-of-concept of larger whole transcriptome studies where all avenues should be used to subtract changes in gene expression due to extrinsic aging from changes potentially due to intrinsic aging.

Placental glutathione S-transferase correlates with cellular proliferation during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

Taking into consideration that glutatione S-transferase (GST) and cellular proliferation play a crucial role during carcinogenesis, the goal of this study was to investigate the expression of placental GST, called GST-P, and proliferating cellular nuclear antigen (PCNA) by means of immunohistochemistry during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide (4NQO). This is a useful model for studying oral squamous cell carcinoma phase by phase. Male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution by drinking water for 4, 12 or 20 weeks. Ten animals were used as negative control. GST-P positive foci were detected in non-neoplastic oral cells at 4 weeks of 4NQO administration. In the same way, GST-P positive cells were detected in pre-neoplastic lesions and squamous cell carcinomas induced after 12 and 20 weeks-treatment, respectively. None of the control animals expressed GST-P positive cells. Regarding cellular proliferation, PCNA positive nuclei were higher at 12 and 20 weeks following 4NQO exposure ( p<0.05) when compared to negative control. These results suggest that the expression of GST-P is correlated with cellular proliferation, in which GST-P is associated with risk and progression of oral cancer, whereas PCNA is closely involved during neoplastic conversion.

A Mu‐class glutathione S‐transferase from gills of the marine shrimp Litopenaeus vannamei: Purification and characterization

Glutathione S-transferases (GSTs) are a family of detoxifying enzymes that catalyze the conjugation of glutathione (GSH) to electrophiles, thereby increasing the solubility of GSH and aiding its excretion from the cell. In this study, a glutatione S-transferase from the gills of the marine shrimp Litopenaeus vannamei was purified by affinity chromatography using a glutathione-agarose affinity column. GST was purified to homogeneity as judged by reducing SDS-PAGE and zymograms. This enzyme is a homodimer composed of approximately 25-kDa subunits and identified as a Mu-class GST based on its activity against 1-chloro-2,4-dinitrobenzene (CDNB) and internal peptide sequence. The specific activity of purified GST was 440.12 micromol/(min mg), and the K(m) values for CDNB and GSH are very similar (390 and 335 microM, respectively). The intersecting pattern of the initial velocities of this enzyme in the Lineweaver-Burke plot is consistent with a sequential steady-state kinetic mechanism. The high specific activity of shrimp GST may be related to a highly effective detoxification mechanism necessary in gills since they are exposed to the external and frequently contaminated environment.

Mechanism of Radiosensitization by Porphyrins

According to our previous data, hematoporphyrin dimethyl ether (HPde) at concentrations useful for photodynamic therapy can radiosensitize aggressive Ehrlich ascite carcinoma (EAT) to 2Gy irradiation inducing total tumour growth inhibition. The aim of this study was to further investigate the possible mechanism of radiosensitization of EAT by dicarboxylic porphyrin-HPde. Our results reveal that HPde is inducing several rearrangements in the EAT cells: 1.2 x 10-6 M of the photosensitizer diminishes the number of cells in mitosis by a factor of 3, increases the number of cells in the S phase of the cell cycle, modifies the activities of antioxidant enzymes glutation S-transferase (GST) and DT-diaphorase (DTD), and eventually induces slight apoptosis. Moreover, it was shown that HPde is a ligand of peripheral benzodiazepine receptor (PBR). Named "house keeper," PBR is usually responsible for all these perturbations, which, in our case, act in concert with the following ionizing radiation, producing the interaction of two antiproliferative/destructive factors.

Effect of curcumin and 4-n-propil curcumin on glutation s-transferase activity in rat liver

Curcumin, a plant phenol in rhizome Curcuma longa L. has been reported to be a potent inhibitor of glutathione S-transferase (GST) in rat liver with in vitro with substrate 1,2-dikloro-4-nitrobenzen (DCNB). By using DCNB, a specific substrate for GST mu, This work was aimed to know whether curcumin derivatives: 4-n-propyl curcumin can also inhibit GST activity in vitro. Glutathione activity can be measured by conjugating GSH and DCNB catalized with GST. The product can be measured spectrofotometrically. Curcumin and its derivatives decreased GST activities with IC50 values 6,86 μM for curcumin and 8,41 μM for 4-n-propyl curcumin. This means that 4-n-propyl curcumin has a weaker inhibition potency than curcumin. Curcumin (consentration 10μM) inhibited GST activity by mixed with the Ki dan Ki’ values 2,77 μM dan 7,33 μM, whereas 4-n-propyl curcumin (consentration 10μM) inhibited GST activity by competitive with the Ki values 6,28 μM. Key words : Curcumin, 4-n-propyl curcumin, glutathione S-transferase, 1,2-dikloro-4-nitrobenzen.

Polymorphism of Human Glutatione S-Transferase Gene in the Populations of the Volga-Ural Region

The frequency of the GstM1 gene deletion homozygotes in eight populations of the Volga–Ural region belonging according to linguistic classification to Turkic (Bashkirs, Tatars, and Chuvashs), Finno–Ugric (Maris, Komis, Mordovians, and Udmurts), and Eastern–Slavic (Russians) ethnic groups, was examined by means of PCR technique. The frequency of the deletion homozygotes varied from 41.4% in Bashkirs to 61.3% in Mordovians. The mean deletion frequency comprised 50.1%, which was consistent with the data for European populations (χ2= 0.009).

Biomarkers for exposure to ambient air pollution--comparison of carcinogen-DNA adduct levels with other exposure markers and markers for oxidative stress.

Human exposure to genotoxic compounds present in ambient air has been studied using selected biomarkers in nonsmoking Danish bus drivers and postal workers. A large interindividual variation in biomarker levels was observed. Significantly higher levels of bulky carcinogen-DNA adducts (75.42 adducts/10(8) nucleotides) and of 2-amino-apidic semialdehyde (AAS) in plasma proteins (56.7 pmol/mg protein) were observed in bus drivers working in the central part of Copenhagen, Denmark. In contrast, significantly higher levels of AAS in hemoglobin (55.8 pmol/mg protein), malondialdehyde in plasma (0. 96 nmol/ml plasma), and polycyclic aromatic hydrocarbon (PAH)-albumin adduct (3.38 fmol/ microg albumin) were observed in the suburban group. The biomarker levels in postal workers were similar to the levels in suburban bus drivers. In the combined group of bus drivers and postal workers, negative correlations were observed between bulky carcinogen-DNA adduct and PAH-albumin levels (p = 0.005), and between DNA adduct and [gamma]-glutamyl semialdehyde (GGS) in hemoglobin (p = 0.11). Highly significant correlations were found between PAH-albumin adducts and AAS in plasma (p = 0.001) and GGS in hemoglobin (p = 0.001). Significant correlations were also observed between urinary 8-oxo-7, 8-dihydro-2'-deoxyguanosine and AAS in plasma (p = 0.001) and PAH-albumin adducts (p = 0.002). The influence of the glutatione S-transferase (GST) M1 deletion on the correlation between the biomarkers was studied in the combined group. A significant negative correlation was only observed between bulky carcinogen-DNA adducts and PAH-albumin adducts (p = 0.02) and between DNA adduct and urinary mutagenic activity (p = 0.02) in the GSTM1 null group, but not in the workers who were homozygotes or heterozygotes for GSTM1. Our results indicate that some of the selected biomarkers can be used to distinguish between high and low exposure to environmental genotoxins.

Glutathione S-transferase M1 (GSTM1) and T1 (GSTT1) genetic polymorphism and susceptibility to gastric and colorectal adenocarcinoma.

Genes coding for the glutathione S-transferase M1 (GSTM1) and Theta 1 (GSTT1) proteins are polymorphic in humans and these genes are absent, or homozygous null, in 10-60% of different ethnic populations. These enzymes catalyze the conjugation of glutathione to numerous carcinogenic chemicals and previous epidemiologic studies have associated the null genotypes of these GST genes with higher risk of cancer. In this study the frequency of GSTM1 and GSTT1 null genotypes was determined in Japanese patients with gastric adenocarcinoma and colorectal adenocarcinoma and compared to frequencies determined in a community-based control group. The frequency of the null GSTM1 genotype in patients with gastric adenocarcinoma (56.8%) showed a statistically significant increase compared to the control group frequency (43.6%) (odds ratio (OR) = 1.70; 95% CI, 1.05-2.76). The frequency of GSTM1 null individuals was also higher among all colorectal adenocarcinoma cases, but this increase did not reach statistical significance. After grouping by tumor site, the GSTM1 null genotype was a risk factor among the subgroup with distal colorectal tumors (61.1%) (OR = 2.03; 95% CI, 1.06-3.90). No consistent difference was observed between smoking patients and corresponding controls for the frequency of the GSTM1 null genotype for either cancer, although a large risk (OR = 5.76; 95% CI 1.18-28.3) was associated with the GSTM1 null genotype in the low smoking group of gastric adenocarcinoma patients. On the other hand, no statistically significant differences were observed in the frequency of null GSTT1 genotypes in gastric (47.5%) or colorectal (48.5%) adenocarcinoma patients when compared with the control population (44.4%). These results suggest that the GSTM1 null genotype may be associated with susceptibility to gastric adenocarcinoma and distal colorectal adenocarcinoma in Japanese; however, the associations observed were relatively weak and additional studies will be needed to confirm these findings.

Characterization of two conformational epitopes of the Chlamydia trachomatis serovar L2 DnaK immunogen.

Chlamydia trachomatis DnaK is an important immunogen in chlamydial infections. DnaK is composed of a conserved N-terminal ATP-binding domain and a variable C-terminal peptide-binding domain. To locate the immunogenic part of C. trachomatis Dnak, we generated monoclonal antibodies (MAbs) against this protein. By use of recombinant DNA techniques, we located the epitopes for two MAbs in the C-terminal variable part. Although the antibodies reacted in an immunoblot assay, it was not possible to map the epitopes completely by use of 16-mer synthetic peptides displaced by one amino acid corresponding to the C-terminal part of C. trachomatis DnaK. To determine the limits of the epitopes, C. trachomatis DnaK and glutatione S-transferase fusion proteins were constructed and affinity purified. The purified DnaK fusion proteins were used for a fluid-phase inhibition enzyme-linked immunosorbent assay with the two antibodies. The epitopes were found not to overlap. To obtain DnaK fragments recognized by the antibodies with the same affinity as native C. trachomatis DnaK, it was necessary to express, respectively, regions of 127 and 77 amino acids. The MAbs described in this study thus recognized conformational epitopes of C. trachomatis DnaK.

Reversible Immunoprecipitation Using Histidine- or Glutathione S-Transferase-Tagged Staphylococcal Protein A

We have constructed, expressed, and purified hexahistidine- and glutatione S-transferase (GST)-tagged Staphylococcal protein A. The histidine-tagged protein A bound efficiently to iminodiacetic acid (IDA)-Sepharose loaded with Zn 2+, and the GST-protein A was efficiently retained by glutathione-Sepharose. Both recombinant forms of protein A can be used in the normal way to harvest immune complexes with IgG. Both forms of protein A can be released from the Sepharose matrix by mild procedures. The his 6-protein A:antibody:antigen complexes can be released from the matrix with EDTA, and immunoprecipitates bound to GST-protein A can be released either by elution with glutathione or by digestion with thrombin. We tested this method with immunoprecipitates of the p40 MO15 protein kinase, and found that they retained their ability to phosphorylate p33 cdk2 after elution from the affinity matrices.

Induction of Glutatione S-Transferase P-Form in Rat Hepatocytes by Coplanar Polychlorinated Biphenyl Congeners and Its Mechanism (Proceedings of the 18th Symposium on Toxicology, and Environmental Health)

Induction of Glutatione S-Transferase P-Form in Rat Hepatocytes by Coplanar Polychlorinated Biphenyl Congeners and Its Mechanism (Proceedings of the 18th Symposium on Toxicology, and Environmental Health)

Conjugation of 2-chloroacetanilide herbicides with glutathione: role of molecular structures and of glutathione S-transferase enzymes

The congugation of acetochlor, alachlor, butachlor, dimetachlor, metolachlor, pretilachlor, and propachlor with glutatione nonenzymatically and enzymatically by action of the glutatione S-transferase (GST) was investigated. Shoots of corn (Zea mays), soybean (Glycine max), wheat (Triticum aestivum), sorghum (Sorghum bicolor), redroot pigweed (Amaranthus retroflexus), and lambsquarter (Chenopodium album) were employed as enzyme sources for enzymatic tests (...)

The enhancing effect of ethanol on the development of glutatione S-transferase placental form-positive foci induced by diethylnitrosamine in F344 rat

The effects of ethyl alcohol and pig serum administration on the development of preneoplastic hepatic enzyme-altered foci were examined in an in vivo mid-term assay system. Rats were initially given a single dose (200 mg/Kg) intraperitoneal injection of diethylnitrosamine (DEN). Two weeks later, treatment was started with 10% ethanol + 10% sucrose solution, 10% sucrose solution, or tap water as drinking water for 6 weeks with or without intraperitoneal injection of porcine serum twice a week. All rats were subjected to a two-thirds partial hepatectomy at week 3. The modification potentials were evaluated by comparing the number and area per cm2 of glutathione S-transferase placental form-positive (GST-P+) foci in the liver of each group. As a result, ethanol significantly enhanced the development of GST-P+ foci. Unfortunately, the porcine serum injection produced no hepatic fibrosis and no significant alteration in GST-P+ foci.

Halothane: Inhibition and activation of rat hepatic glutathione s-transferases

Multiple halothane anesthesias (1.25 MAC for 1 hr on 3 alternate days) of male Long-Evans rats initially decreased by up to 30% and subsequently increased to up to 185% liver cytosolie glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene, 3,4-dichloro-1-nitrobenzene and trans-4-phenyl-3-buten-2-one and glutathione peroxidase activity. Halothane rapidly and reversibly activated hepatic cytosolie glutathione S-transferases and purified isoenzyme 1-2 but not isoenzymes 1-1 and 3-3. At high concentrations of halothane ( ca. 22 mM), maximal activation was co. 25%. Halothane, enflurane, isoflurane and methoxyflurane, but not the halothane metabolite 1-chloro-2,2-difluoroethylene, inhibited a mixture of liver cytosolie glutathione S-transferases with time ( ca. 30% inhibition/15 min). The inhibition exhibited pseudo-first order kinetics ( k obc = 0.13 min − and an I 50 for halothane of ⩾15 mM. Halothane inhibited glutathione S-transferases 3-3,3-4, and 4-4 by 50–60%, but did not affect isoenzymes 1-1 and 1-2. The ability of halothane to diminish hepatic glutatione S-transferase activity in vivo may in part reflect the time-dependent inhibition of glutathione S-transferase isoenzymes containing the 3-and 4-subunits.

An organic peroxide, methyl ethyl ketone peroxide, damage to cytochrome P-450 peroxidase and microsomal enzymes activity

In vivo, an organic peroxide, methyl ethyl ketone peroxide (MEKP) damaged microsomal cytochrome P-450 and cytochrome P-450 peroxidase in vitamin E deficient rat livers. Dietary vitamin E protected the microsomal enzymes from MEKP damage. Phenobarbital induced liver cytochrome P-450 peroxidase and glutation S-transferase and decreased MEKP damage on microsomal enzymes. MEKP induced production of more lipid peroxides (TBARS) in liver microsomes from vitamin E deficient rats than from vitamin E supplemented rats. Cytochrome P-450 and aminopyrine demethylase from vitamin E deficient rat microsomes were damaged to a greater extent by MEKP than were those from vitamin E supplemented rat microsomes. MEKP markedly inhibited liver microsomal TMPD- and NADH-peroxidase. In vivo, adequate levels of vitamin E, NADH, and NADPH are probably necessary to provide important protection to the endoplasmic reticulum from damage by organic peroxides.